Fig. 3

Biochemical assays of two sugar transfers to Rubu by UGT76G1. a HPLC traces of the reactions of Rubu. The five HPLC traces from top to bottom represent the same reaction conditions as Fig. 2. The first product of Rubu is arbitrarily assigned as RX, and the second one is assigned as RY. The yields of the products are related to the enzyme concentration and the reaction duration. The reactions catalyzed by UGT76G1 are shown in the box. b Direct MS of two sugar transfers of Rubu by 0.15 mg ml⁻¹ UGT76G1 (5×) for 2 and 18 h. The two main negative ions derived from the products RX and RY are labeled and show the same characteristics in terms of the relative contents of the products as a function of the reaction duration as shown by HPLC (Fig. 3a). c MS/MS of the authentic Rubu standard and the collected HPLC peaks of the products RX and RY. The negative ions [Rubu–H]⁻, [RX–H]⁻, and [RY–H]⁻ with m/z at 641.3, 803.4 and 965.4, respectively, were specifically isolated as the parent ions and characterized by MS/MS. The most labile ester bond breaks first, which was consistently indicated by the abundant fragment ions of Rubu, RX, and RY and was used to identify the positions of the added sugars transferred by UGT76G1. The inlet suggests where the ester bond breaks first during MS/MS fragmentation